JP2012102041A - Antioxidant and anti-inflammatory agent - Google Patents
Antioxidant and anti-inflammatory agent Download PDFInfo
- Publication number
- JP2012102041A JP2012102041A JP2010251333A JP2010251333A JP2012102041A JP 2012102041 A JP2012102041 A JP 2012102041A JP 2010251333 A JP2010251333 A JP 2010251333A JP 2010251333 A JP2010251333 A JP 2010251333A JP 2012102041 A JP2012102041 A JP 2012102041A
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- inhibitor
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- davidigenin
- hydrogen peroxide
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Abstract
Description
本発明は、過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤、抗炎症剤、及びダビジゲニンの製造方法に関するものである。 The present invention relates to a preventive / ameliorating agent for hydrogen peroxide cell damage, a glutathione production promoter, an antioxidant, a tumor necrosis factor (TNF-α) production inhibitor, a hexosaminidase release inhibitor, a cyclooxygenase-2 inhibitor, The present invention relates to an anti-inflammatory agent and a method for producing davidigenin.
近年、生体成分を酸化させる要因として活性酸素が注目されており、生体への悪影響が問題となっている。活性酸素は、主に生体細胞内におけるエネルギー代謝過程で生じるものであり、スーパーオキサイド（すなわち酸素分子の一電子還元で生じるスーパーオキシドアニオン：・Ｏ２ −）、過酸化水素（Ｈ２Ｏ２）、ヒドロキシラジカル（・ＯＨ）及び一重項酸素（１Ｏ２）などがある。これらの活性酸素は、好中球やマクロファージなどの食細胞による細胞内殺菌機構に関与するものであり、ウイルスや癌細胞の除去に重要な役割を果たしているが、活性酸素が過剰に生成されると、活性酸素が細胞膜や組織を構成する生体内分子を攻撃し、各種疾患を誘発するおそれがある。 In recent years, active oxygen has attracted attention as a factor that oxidizes biological components, and adverse effects on living organisms have become a problem. Active oxygen is mainly generated in the process of energy metabolism in living cells, and is superoxide (that is, superoxide anion generated by one-electron reduction of oxygen molecules: .O 2 − ), hydrogen peroxide (H 2 O 2 ). , Hydroxy radical (.OH) and singlet oxygen ( 1 O 2 ). These active oxygens are involved in the intracellular sterilization mechanism by phagocytic cells such as neutrophils and macrophages and play an important role in the removal of viruses and cancer cells, but excessive oxygen is generated Then, active oxygen may attack in vivo molecules constituting cell membranes and tissues and induce various diseases.
例えば、過酸化水素は、炎症、過酸化脂質の生成、種々のタンパク質の変性・失活及びＤＮＡの損傷などを引き起こすことが知られており、これらが原因となって誘発される疾患として、喫煙等が原因の肺疾患、白内障、各種動脈硬化症（虚血性心疾患、心筋梗塞、脳虚血、脳梗塞等）、加齢に伴う老化現象、神経変性疾患（アルツハイマー病、パーキンソン病、ハンチントン舞踏病等）、癌化等が知られている。 For example, hydrogen peroxide is known to cause inflammation, production of lipid peroxides, denaturation / inactivation of various proteins, and DNA damage. Pulmonary diseases, cataracts, various arteriosclerosis (ischemic heart disease, myocardial infarction, cerebral ischemia, cerebral infarction, etc.), aging with age, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's choreography) Disease, etc.), canceration, etc. are known.
そのため、過酸化水素によって生じる細胞障害を予防・改善して、細胞の恒常性を高めることができれば、過酸化水素による酸化ストレスが原因となって誘発される上記の疾患群を予防・改善できるものと考えられる。このような過酸化水素細胞障害の予防・改善作用を有するものとして、五斂子花部の抽出物（特許文献３参照）等が知られている。 Therefore, if the cell damage caused by hydrogen peroxide can be prevented / improved and the homeostasis of the cells can be increased, the above-mentioned diseases caused by oxidative stress caused by hydrogen peroxide can be prevented / improved it is conceivable that. As a substance having such an action of preventing or improving hydrogen peroxide cell damage, an extract of a quince flower part (see Patent Document 3) and the like are known.
また、グルタチオンは、グルタミン酸、システイン及びグリシンの３つのアミノ酸からなるトリペプチドであり、細胞内の主要なシステイン残基を有する化合物である。細胞内におけるグルタチオンは、ラジカルの捕捉、酸化還元による細胞機能の調節、異物代謝、各種酵素のＳＨ供与体としての機能を果たすものであり、抗酸化成分としても知られている。その作用発現は、システイン残基に由来すると考えられている。しかしながら、過剰な酸化ストレスや異物の付加、加齢などにより、細胞内のグルタチオン量が欠乏又は低下することが報告されており、このことが細胞の酸化ストレスに対する防御能を低下させ、細胞のＤＮＡ及びタンパク質等の構成成分にダメージを与える一因であると考えられている。 Glutathione is a tripeptide composed of three amino acids, glutamic acid, cysteine and glycine, and is a compound having a major cysteine residue in the cell. Glutathione in cells functions as a radical donor, regulation of cell function by redox, foreign body metabolism, SH donor of various enzymes, and is also known as an antioxidant component. Its action expression is thought to be derived from cysteine residues. However, it has been reported that the amount of glutathione in cells is deficient or decreased due to excessive oxidative stress, addition of foreign substances, aging, etc., and this reduces the protective ability of cells against oxidative stress, and the cellular DNA In addition, it is considered to be a cause of damaging components such as proteins.
このような、細胞内のグルタチオン量の低下又は欠乏が病態と関連することが知られている疾患として、酸化ストレスが原因となって誘発される上記の疾患群のほか、肝障害（アルコールの多飲、又は重金属や化学物質等の異物の摂取が原因となる）等が知られている。 In addition to the above-mentioned diseases that are induced by oxidative stress, diseases such as a decrease or deficiency in the amount of intracellular glutathione are known to be associated with the disease state. It is caused by drinking or ingestion of foreign substances such as heavy metals and chemical substances).
すなわち、グルタチオンの産生を促進することは、細胞の酸化ストレスに対する防御能を高め、細胞内のグルタチオン量が低下又は欠乏することに起因する上記の疾患群を予防・治療することができると考えられる。このようなグルタチオン産生促進作用を有するものとして、テンニンカ抽出物（特許文献１参照）、クチナシ属植物の抽出物（特許文献２参照）等が知られている。 That is, it is considered that promoting the production of glutathione can enhance the ability of cells to protect against oxidative stress, and can prevent and treat the above-mentioned disease group caused by the decrease or lack of intracellular glutathione level. . As those having such a glutathione production promoting action, a tenninka extract (see Patent Document 1), an extract of a gardenia plant (see Patent Document 2), and the like are known.
炎症性疾患、例えば、接触性皮膚炎（かぶれ）、乾癬、尋常性天疱瘡、アトピー性皮膚炎、その他肌荒れを伴う各種皮膚疾患、関節リウマチ、変形性関節症、喘息等の原因及び発症機構は、多種多様である。その原因として、主にマクロファージから産生される腫瘍壊死因子（以下「ＴＮＦ−α」と称することもある。）によるもの、ヒスタミンの遊離によるもの、及びシクロオキシゲナーゼ−２の活性の亢進によるもの等が知られている。 Causes and onset mechanisms of inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, atopic dermatitis, other skin diseases with rough skin, rheumatoid arthritis, osteoarthritis, asthma, etc. A wide variety. Known causes include tumor necrosis factor (hereinafter sometimes referred to as “TNF-α”) produced mainly by macrophages, histamine release, and cyclooxygenase-2 activity enhancement. It has been.
ＴＮＦ−αは、腫瘍を壊死させる因子として見出されたが、最近では腫瘍に対してだけでなく、正常細胞の機能を調節するメディエーター的な役割を担うサイトカインであると考えられている。ＴＮＦ−αは、炎症の初発から終息までの過程において重要な役割を担っているが、その持続的かつ過剰な産生は、皮膚を含む組織の障害を引き起こし、全身的には発熱やカケクシアの原因となり、炎症の悪化を引き起こす。したがって、病的な炎症においては、ＴＮＦ−αの過剰な産生を抑制することが重要となる。このようなＴＮＦ−α産生抑制作用を有するものとして、例えば、土貝母抽出液（特許文献３参照）、マルツロシルアルギニン及びフルクトシルアルギニン（特許文献４参照）等が知られている。 TNF-α was found as a factor that necrotizes tumors, but is recently considered to be a cytokine that plays a mediator role not only for tumors but also for regulating the function of normal cells. TNF-α plays an important role in the process from the onset to the end of inflammation, but its persistent and excessive production causes damage to tissues including the skin, causing systemic fever and cyccia. And cause worsening of inflammation. Therefore, in pathological inflammation, it is important to suppress excessive production of TNF-α. As what has such a TNF- (alpha) production inhibitory effect, for example, a shellfish mother extract (refer patent document 3), a marturosyl arginine, a fructosyl arginine (refer patent document 4), etc. are known.
ヒスタミン遊離は、肥満細胞内のヒスタミンが細胞外に遊離する現象であり、遊離されたヒスタミンが炎症反応を引き起こす。そのため、ヒスタミン遊離を阻害又は抑制する物質により、アレルギー性疾患及び炎症性疾患を予防又は治療する試みがなされている。しかし、ヒスタミンの遊離を直接的に評価することは困難であり、ヒスタミンの遊離と同時に遊離されることが確認されているヘキソサミニダーゼの遊離を指標にヒスタミンの遊離を評価することができる。したがって、ヘキソサミニダーゼの遊離を抑制することにより、同時にヒスタミンの遊離も抑制でき、これにより炎症性疾患等の予防、治療又は改善に効果があるものと考えられる。このようなヘキソサミニダーゼ遊離抑制作用を有する植物抽出物としては、藤茶からの抽出物（特許文献５参照）等が知られている。 Histamine release is a phenomenon in which histamine in mast cells is released extracellularly, and the released histamine causes an inflammatory reaction. Therefore, attempts have been made to prevent or treat allergic diseases and inflammatory diseases with substances that inhibit or suppress histamine release. However, it is difficult to directly evaluate the release of histamine, and the release of histamine can be evaluated using as an index the release of hexosaminidase that has been confirmed to be released simultaneously with the release of histamine. Therefore, it is considered that by suppressing the release of hexosaminidase, the release of histamine can also be suppressed at the same time, which is effective for preventing, treating or improving inflammatory diseases. As a plant extract having such a hexosaminidase release inhibitory effect, an extract from Fuji tea (see Patent Document 5) and the like are known.
また、炎症は、発赤、浮腫、発熱、痛み、機能障害等の症状を示す複雑な反応である。微視的に見ると、炎症は、血漿漏出を起こす血管反応、白血球の浸潤、炎症性細胞による組織破壊等の共通する反応からなり、発熱反応や痛覚過敏等の中枢神経系も関与する、全身反応も引き起こす場合もある。このような炎症の個々の反応には、プロスタグランジンが重要な役割を果たしており、この炎症時におけるプロスタグランジンの産生には、主として誘導型のシクロオキシゲナーゼであるシクロオキシゲナーゼ−２が関与することが明らかとなっている。 Inflammation is a complex reaction that exhibits symptoms such as redness, edema, fever, pain, and dysfunction. Microscopically, inflammation consists of common reactions such as vascular reactions that cause plasma leakage, leukocyte infiltration, tissue destruction by inflammatory cells, and the central nervous system such as fever and hyperalgesia is also involved. It may also cause a reaction. It is clear that prostaglandins play an important role in each reaction of such inflammation, and that cyclosogenase-2, which is an inducible cyclooxygenase, is mainly involved in the production of prostaglandins during this inflammation. It has become.
このため、炎症反応の防止及び予防を図る目的で、アスピリンに代表される多くのシクロオキシゲナーゼ阻害剤が用いられている（非特許文献１参照）。また、シクロオキシゲナーゼ阻害作用を有する植物抽出物としては、バーベリー抽出物等が知られている（特許文献６参照）。また、シクロオキシゲナーゼ−２活性阻害作用を有する化合物としては、２−フェニル−１，２−ベンズイソセレナゾール−３（２Ｈ）−オン、その塩又はその水和物等が知られている（特許文献７参照）。 For this reason, many cyclooxygenase inhibitors represented by aspirin are used for the purpose of preventing and preventing inflammatory reactions (see Non-Patent Document 1). Moreover, a barberry extract etc. are known as a plant extract which has a cyclooxygenase inhibitory effect (refer patent document 6). Further, as a compound having an inhibitory action on cyclooxygenase-2 activity, 2-phenyl-1,2-benzisoselenazol-3 (2H) -one, a salt thereof, a hydrate thereof, or the like is known (Patent Literature). 7).
しかしながら、優れた過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用又は抗炎症作用を有し、安価である過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤に対する消費者の要望は強く、さらなる新しい過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤の開発及び提供が強く求められているのが現状である。 However, excellent hydrogen peroxide cell damage prevention / amelioration action, glutathione production promoting action, antioxidant action, tumor necrosis factor (TNF-α) production inhibiting action, hexosaminidase release inhibiting action, cyclooxygenase-2 inhibiting action or Anti-inflammatory and inexpensive hydrogen peroxide cell damage preventive / ameliorating agent, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, Consumer demand for cyclooxygenase-2 inhibitors or anti-inflammatory agents is strong, further new hydrogen peroxide cell damage prevention / amelioration agents, glutathione production promoters, antioxidants, tumor necrosis factor (TNF-α) production inhibitors The development and provision of a hexosaminidase release inhibitor, a cyclooxygenase-2 inhibitor or an anti-inflammatory agent is currently strongly demanded. It is.
本発明は、優れた過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用又は抗炎症作用を有する化合物を見出し、それを有効成分とする過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤を提供することを目的とする。 The present invention has an excellent action for preventing and improving hydrogen peroxide cell damage, glutathione production promoting action, antioxidant action, tumor necrosis factor (TNF-α) production inhibiting action, hexosaminidase release inhibiting action, cyclooxygenase-2 inhibition A compound having an action or an anti-inflammatory action, and an agent for preventing or improving hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, It aims at providing a sosaminidase release inhibitor, a cyclooxygenase-2 inhibitor, or an anti-inflammatory agent.
上記課題を解決するために、本発明の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、ダビジゲニンを有効成分として含有することを特徴とする。 In order to solve the above-mentioned problems, the preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor of the present invention The cyclooxygenase-2 inhibitor or anti-inflammatory agent contains davidigenin as an active ingredient.
本発明によれば、過酸化水素細胞障害の予防・改善作用に優れた過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進作用に優れたグルタチオン産生促進剤、抗酸化作用に優れた抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用に優れた腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制作用に優れたヘキソサミニダーゼ遊離抑制剤、又はシクロオキシゲナーゼ−２阻害作用に優れたシクロオキシゲナーゼ−２阻害剤、抗炎症作用に優れた抗炎症剤を提供することができる。 According to the present invention, a hydrogen peroxide cell disorder preventive / ameliorating agent excellent in hydrogen peroxide cell disorder preventing / ameliorating action, a glutathione production promoter excellent in glutathione production promoting action, and an antioxidant excellent in antioxidant action Agent, tumor necrosis factor (TNF-α) production inhibitor excellent in tumor necrosis factor (TNF-α) production inhibitory activity, hexosaminidase release inhibitor excellent in hexosaminidase release inhibitory activity, or cyclooxygenase- 2 It is possible to provide a cyclooxygenase-2 inhibitor excellent in an inhibitory action and an anti-inflammatory agent excellent in an anti-inflammatory action.
以下、本発明の実施の形態について説明する。
本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、下記式で表されるダビジゲニンを有効成分として含有するものである。
Embodiments of the present invention will be described below.
The preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor or The anti-inflammatory agent contains davidigenin represented by the following formula as an active ingredient.
ダビジゲニンは、合成により製造することもできるし、ダビジゲニンを含有する植物の抽出物から単離・精製することにより製造することもできる。さらに、２’，４，４’−トリヒドロキシカルコン（イソリクイリチゲニン）を含有する植物の抽出物を還元処理し、当該抽出物に含まれる２’，４，４’−トリヒドロキシカルコンをダビジゲニンに還元した後、単離・精製することにより製造することも可能である。 Davidigenin can be produced synthetically, or can be produced by isolation and purification from an extract of a plant containing davidigenin. Further, a plant extract containing 2 ′, 4,4′-trihydroxychalcone (isoliqueritigenin) is reduced, and 2 ′, 4,4′-trihydroxychalcone contained in the extract is reduced. It can also be produced by reducing to davidigenin followed by isolation and purification.
ダビジゲニンを合成により製造する場合、公知の方法により合成することができる。例えば、ｐ−ヒドロキシベンズアルデヒドと２，４−ジヒドロキシアセトフェノンとを塩基の存在化でアルドール縮合させ、２’，４，４’−トリヒドロキシカルコンを得る。得られた２’，４，４’−トリヒドロキシカルコンにさらに水素を添加することによりダビジゲニンを得ることができる。 When davidigenin is produced by synthesis, it can be synthesized by a known method. For example, p-hydroxybenzaldehyde and 2,4-dihydroxyacetophenone are subjected to aldol condensation in the presence of a base to obtain 2 ', 4,4'-trihydroxychalcone. By further adding hydrogen to the obtained 2 ', 4,4'-trihydroxychalcone, davidigenin can be obtained.
アルドール縮合反応において使用し得る溶媒としては、水；メタノール、エタノール、プロパノール等のアルコール；ｎ−ヘキサン、ベンゼン等の炭化水素系有機溶媒；ジメチルスルホキシド等が挙げられ、これらを１種又は２種以上の混合溶媒として使用することができる。また、アルドール縮合において触媒として使用し得る塩基としては、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、炭酸カリウム、ナトリウムメチラート、ナトリウムエチラート等が挙げられる。これらの塩基は、適宜溶媒に溶解して加えても良い。アルドール縮合における反応温度は−１０〜８０℃であることが好ましい。反応後、再結晶などの一般的な精製手法により、２’，４，４’−トリヒドロキシカルコンを得ることができる。 Solvents that can be used in the aldol condensation reaction include water; alcohols such as methanol, ethanol, and propanol; hydrocarbon-based organic solvents such as n-hexane and benzene; dimethyl sulfoxide, and the like. It can be used as a mixed solvent. Examples of the base that can be used as a catalyst in the aldol condensation include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium methylate, sodium ethylate and the like. These bases may be added by appropriately dissolving in a solvent. The reaction temperature in the aldol condensation is preferably -10 to 80 ° C. After the reaction, 2 ', 4,4'-trihydroxychalcone can be obtained by a general purification technique such as recrystallization.
このようにして得られた２’，４，４’−トリヒドロキシカルコンに水素を添加する方法としては、接触還元を用いることができる。接触還元において使用する溶媒としては、水；メタノール、エタノール、プロパノール等のアルコール；酢酸エチル、ジエチルエーテル、ジオキサン等が挙げられ、これらを１種又は２種以上の混合溶媒として使用することができる。接触還元において使用し得る触媒としては、パラジウム、パラジウムカーボン、白金、酸化白金などが挙げられる。接触還元における反応温度は−１０〜８０℃であることが好ましい。反応後、再結晶などの一般的な精製手法により、ダビジゲニンを得ることができる。 Catalytic reduction can be used as a method for adding hydrogen to the 2 ', 4,4'-trihydroxychalcone thus obtained. Examples of the solvent used in the catalytic reduction include water; alcohols such as methanol, ethanol, and propanol; ethyl acetate, diethyl ether, dioxane, and the like, and these can be used as one or more mixed solvents. Examples of the catalyst that can be used in the catalytic reduction include palladium, palladium carbon, platinum, platinum oxide and the like. The reaction temperature in the catalytic reduction is preferably −10 to 80 ° C. After the reaction, davidigenin can be obtained by a general purification technique such as recrystallization.
なお、上記の方法において、ｐ−ヒドロキシベンズアルデヒドと２，４−ジヒドロキシアセトフェノンとに代えて、これらの化合物に存在する１又は２以上のヒドロキシル基を保護基によりあらかじめ保護した化合物を用いてアルドール縮合を行い、得られた縮合物を還元する前又は還元した後に脱保護を行うことにより、ダビジゲニンを得ることとしても良い。 In the above method, aldol condensation is performed using a compound in which one or two or more hydroxyl groups present in these compounds are protected in advance by a protecting group instead of p-hydroxybenzaldehyde and 2,4-dihydroxyacetophenone. It is also possible to obtain davidigenin by deprotection before or after reducing the resulting condensate.
また、２’，４，４’−トリヒドロキシカルコンを含有する植物の抽出物を還元処理し、２’，４，４’−トリヒドロキシカルコンをダビジゲニンに還元した後、単離・精製することにより製造する場合、以下の方法により製造することができる。この方法により、後述する好ましい生理作用を有するダビジゲニンを、天然物から安価にかつ大量に製造することが可能となる。 In addition, a plant extract containing 2 ′, 4,4′-trihydroxychalcone is reduced, 2 ′, 4,4′-trihydroxychalcone is reduced to davidigenin, and then isolated and purified. When manufacturing, it can manufacture by the following methods. By this method, davidigenin having a preferable physiological action described later can be produced at low cost and in large quantities from a natural product.
２’，４，４’−トリヒドロキシカルコンを含有する植物としては、例えば、甘草（Glychyrrhiza属）の植物が挙げられる。２’，４，４’−トリヒドロキシカルコンを含有する植物抽出物は、植物の抽出に一般に用いられている方法によって得ることができる。例えば、抽出原料を乾燥した後、そのまま又は粗砕機を用いて粉砕し、抽出溶媒による抽出に供することにより得ることができる。抽出溶媒としては、極性溶媒を使用するのが好ましく、例えば、水、親水性有機溶媒等が挙げられ、これらを単独で又は２種以上を組み合わせて、室温又は溶媒の沸点以下の温度で使用することが好ましい。 Examples of plants containing 2 ', 4,4'-trihydroxychalcone include licorice (Glychyrrhiza genus) plants. Plant extracts containing 2 ', 4,4'-trihydroxychalcone can be obtained by methods commonly used for plant extraction. For example, it can be obtained by drying the extraction raw material, pulverizing the raw material as it is or using a crusher, and subjecting it to extraction with an extraction solvent. As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. It is preferable.
抽出処理は、抽出原料に含まれる可溶性成分を抽出溶媒に溶出させ得る限り特に限定はされず、常法に従って行うことができる。例えば、抽出溶媒に抽出原料を浸漬し、常温又は還流加熱下で可溶性成分を抽出させた後、濾過して抽出残渣を除去することにより抽出液を得ることができ、得られた抽出液から溶媒を留去すると濃縮物が、さらに乾燥すると乾燥物が得られる。 The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, an extraction raw material can be obtained by immersing an extraction raw material in an extraction solvent, extracting a soluble component at room temperature or under reflux, and then filtering to remove an extraction residue. When the solvent is distilled off, a concentrate is obtained, and when dried, a dried product is obtained.
以上のようにして得られた抽出液、当該抽出液の濃縮物又は当該抽出液の乾燥物に還元処理を行う方法としては、接触還元を用いることができる。接触還元において使用する溶媒としては、水；メタノール、エタノール、プロパノール等のアルコール；酢酸エチル、ジエチルエーテル、ジオキサン等が挙げられ、これらを単独で又は２種以上を組み合わせて使用することができる。接触還元において使用し得る触媒としては、パラジウム、パラジウムカーボン、白金、酸化白金などが挙げられ、中でもパラジウムカーボンが好ましい。また、接触還元における反応温度は−１０〜８０℃であることが好ましい。 Catalytic reduction can be used as a method for performing a reduction treatment on the extract obtained as described above, the concentrate of the extract or the dried product of the extract. Examples of the solvent used in the catalytic reduction include water; alcohols such as methanol, ethanol and propanol; ethyl acetate, diethyl ether and dioxane. These can be used alone or in combination of two or more. Examples of the catalyst that can be used in the catalytic reduction include palladium, palladium carbon, platinum, platinum oxide and the like, and among them, palladium carbon is preferable. Moreover, it is preferable that the reaction temperature in catalytic reduction is -10-80 degreeC.
以上のようにして還元処理を行った抽出物は、そのままダビジゲニンの単離・精製を行えば良い。なお、還元処理の前後に、酸又は塩基等を用いた加水分解処理を行うことで、ダビジゲニンの収量を高めてもよい。ダビジゲニンを単離・精製する方法は、特に限定されるものではなく、常法により行うことができる。例えば、抽出物を、多孔質物質や多孔性樹脂等を用いたカラムクロマトグラフィーに付すことにより、ダビジゲニンを含有する画分として得ることができる。さらに、得られた画分を、ＯＤＳを用いた逆相シリカゲルクロマトグラフィー、再結晶、液−液向流抽出、イオン交換樹脂を用いたカラムクロマトグラフィー等の任意の有機化合物精製手段を用いて精製してもよい。 The extract subjected to the reduction treatment as described above may be isolated and purified as it is. In addition, the yield of davidigenin may be increased by performing a hydrolysis treatment using an acid or a base before and after the reduction treatment. The method for isolating and purifying davidigenin is not particularly limited, and can be performed by a conventional method. For example, the extract can be obtained as a fraction containing davidigenin by subjecting the extract to column chromatography using a porous substance, a porous resin or the like. Furthermore, the obtained fraction is purified using any organic compound purification means such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, and the like. May be.
以上のようにして得られるダビジゲニンは、優れた過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用及び抗炎症作用を有しているため、過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤及び抗炎症剤の有効成分として用いることができる。 The davidigenin obtained as described above is excellent in preventing and improving hydrogen peroxide cell damage, promoting glutathione production, antioxidant, inhibiting tumor necrosis factor (TNF-α) production, and inhibiting hexosaminidase release. Since it has an action, a cyclooxygenase-2 inhibitory action and an anti-inflammatory action, it prevents or ameliorates hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, It can be used as an active ingredient of hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor and anti-inflammatory agent.
本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、ダビジゲニンのみからなるものでもよいし、ダビジゲニンを製剤化したものでもよい。 The preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor or The anti-inflammatory agent may be composed only of davidigenin or may be formulated with davidigenin.
本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、錠剤状、液状等の任意の剤形に製剤化することができる。この際、助剤としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味・矯臭剤等を用いることができる。過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、他の組成物（例えば，皮膚外用剤，飲食品等）に配合して使用することができるほか、軟膏剤、外用液剤、貼付剤等として使用することができる。 The preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor or Anti-inflammatory agents are formulated into powders, granules, tablets, liquids, etc. in any dosage form using pharmaceutically acceptable carriers such as dextrin and cyclodextrin and other optional auxiliaries according to conventional methods. can do. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring agent, and the like can be used. Hydrogen peroxide cell preventive / ameliorating agent, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor or anti-inflammatory agent In addition to being used in other compositions (for example, external preparations for skin, foods and drinks), they can be used as ointments, external liquids, patches and the like.
本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤を製剤化した場合、ダビジゲニンの含有量は、特に限定されるものではなく、目的に応じて適宜設定することができる。 The preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor or When the anti-inflammatory agent is formulated, the content of davidigenin is not particularly limited and can be appropriately set depending on the purpose.
なお、本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、必要に応じて、過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用又は抗炎症作用を有する他の天然抽出物等を、ダビジゲニンとともに配合して有効成分として用いることができる。 In addition, the preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibition of this embodiment Agents or anti-inflammatory agents prevent or improve hydrogen peroxide cell damage, promote glutathione production, antioxidant, suppress tumor necrosis factor (TNF-α) production, and inhibit hexosaminidase release as necessary Other natural extracts having an action, cyclooxygenase-2 inhibitory action or anti-inflammatory action can be blended with davidigenin and used as an active ingredient.
本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤の患者に対する投与方法としては、経皮投与、経口投与等が挙げられるが、疾患の種類に応じて、その予防・治療等に好適な方法を適宜選択すればよい。 The preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor or Examples of the administration method of the anti-inflammatory agent to the patient include transdermal administration, oral administration, and the like, and a suitable method for the prevention / treatment or the like may be appropriately selected depending on the type of the disease.
また、本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤の投与量も、疾患の種類、重症度、患者の個人差、投与方法、投与期間等によって適宜増減すればよい。 Further, the hydrogen peroxide cell disorder preventive / ameliorating agent, glutathione production promoter, antioxidant, tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibition of the present embodiment The dose of the agent or anti-inflammatory agent may be appropriately increased or decreased depending on the type of disease, severity, individual differences among patients, administration method, administration period, and the like.
本実施形態の過酸化水素細胞障害の予防・改善剤は、有効成分であるダビジゲニンが有する過酸化水素細胞障害の予防・改善作用を通じて、過酸化水素の過剰生成が病態と関連していることが知られている疾患等の治療・予防の用途に使用することができる。このような疾患として、喫煙等が原因の肺疾患、白内障、各種動脈硬化症（虚血性心疾患、心筋梗塞、脳虚血、脳梗塞等）、加齢に伴う老化現象、神経変性疾患（アルツハイマー病、パーキンソン病、ハンチントン舞踏病等）、癌化等が挙げられる。ただし、本実施形態の過酸化水素細胞障害の予防・改善剤は、これらの用途以外にも、過酸化水素細胞障害の予防・改善作用を発揮することに意義のあるすべての用途に用いることができる。 The agent for preventing / ameliorating hydrogen peroxide cell damage according to the present embodiment is that excessive production of hydrogen peroxide is associated with a disease state through the action of preventing / ameliorating hydrogen peroxide cell damage of davidigenin, which is an active ingredient. It can be used for treatment / prevention of known diseases. Such diseases include lung diseases caused by smoking, cataracts, various arteriosclerosis (ischemic heart disease, myocardial infarction, cerebral ischemia, cerebral infarction, etc.), aging with age, neurodegenerative diseases (Alzheimer's disease) Disease, Parkinson's disease, Huntington's chorea, etc.) and canceration. However, the hydrogen peroxide cell disorder preventive / ameliorating agent of the present embodiment can be used for all purposes that are meaningful for exerting the hydrogen peroxide cell disorder preventive / ameliorating action in addition to these uses. it can.
本実施形態のグルタチオン産生促進剤は、有効成分であるダビジゲニンが有するグルタチオン産生促進作用を通じて、細胞内グルタチオン量の低下又は欠乏が病態と関連することが知られている疾患等を予防、治療又は改善することができる。このような疾患として、喫煙等が原因の肺疾患、白内障、各種動脈硬化症（虚血性心疾患、心筋梗塞、脳虚血、脳梗塞等）、加齢に伴う老化現象、神経変性疾患（アルツハイマー病、パーキンソン病、ハンチントン舞踏病等）、癌化、肝障害（アルコールの多飲、又は重金属や化学物質等の異物の摂取が原因となる）等が挙げられる。ただし、本実施形態のグルタチオン産生促進剤は、これらの用途以外にも、グルタチオン産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 The glutathione production promoter of the present embodiment prevents, treats or improves a disease or the like in which a decrease or deficiency of intracellular glutathione is known to be associated with a disease state through the glutathione production promoting action of davidigenin, which is an active ingredient. can do. Such diseases include lung diseases caused by smoking, cataracts, various arteriosclerosis (ischemic heart disease, myocardial infarction, cerebral ischemia, cerebral infarction, etc.), aging with age, neurodegenerative diseases (Alzheimer's disease) Disease, Parkinson's disease, Huntington's chorea, etc.), canceration, liver damage (caused by excessive drinking of alcohol or ingestion of foreign substances such as heavy metals and chemical substances). However, the glutathione production promoter of the present embodiment can be used for all purposes other than these uses that are meaningful for exerting the glutathione production promoting action.
本実施形態の抗酸化剤は、ダビジゲニンが有するグルタチオン産生促進作用及び／又は過酸化水素細胞障害の予防・改善作用を通じて、酸化ストレスにより誘発される各種疾患、すなわち過酸化水素の過剰生成又は細胞内グルタチオン量の低下若しくは欠乏が病態と関連することが知られている上記の疾患群を予防・改善することができる。ただし、ダビジゲニンが有する抗酸化作用は、上記作用に基づいて発揮される抗酸化作用に限定されるものではない。 The antioxidant of the present embodiment is various diseases induced by oxidative stress, i.e., excessive production of hydrogen peroxide or intracellular, through the glutathione production promoting action and / or the prevention / amelioration action of hydrogen peroxide cell damage possessed by davidigenin. It is possible to prevent or ameliorate the above-mentioned disease group in which a decrease or deficiency of glutathione is known to be associated with a disease state. However, the antioxidant action of davidigenin is not limited to the antioxidant action exhibited based on the above action.
本実施形態の腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤は、有効成分であるダビジゲニンが有する腫瘍壊死因子（ＴＮＦ−α）産生抑制作用を通じて、接触性皮膚炎（かぶれ）、乾癬、尋常性天疱瘡、アトピー性皮膚炎、その他肌荒れを伴う各種皮膚疾患、関節リウマチ、変形性関節症、喘息等を予防、治療又は改善することができる。ただし、本実施形態の腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤は、これらの用途以外にも、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用を発揮することに意義あるすべての用途に用いることができる。 The tumor tumor necrosis factor (TNF-α) production inhibitor of this embodiment is a contact dermatitis (rash), psoriasis, vulgaris through the tumor necrosis factor (TNF-α) production inhibitory action of davidigenin, which is an active ingredient. It can prevent, treat or improve pemphigus, atopic dermatitis, other various skin diseases with rough skin, rheumatoid arthritis, osteoarthritis, asthma and the like. However, the tumor tumor necrosis factor (TNF-α) production inhibitor of the present embodiment is used for all uses that are meaningful for exerting the tumor necrosis factor (TNF-α) production inhibitory effect in addition to these uses. be able to.
本実施形態のヘキソサミニダーゼ遊離抑制剤は、ダビジゲニンが有するヘキソサミニダーゼ遊離抑制作用を通じて、接触性皮膚炎（かぶれ）、乾癬、尋常性天疱瘡、アトピー性皮膚炎、その他肌荒れを伴う各種皮膚疾患、関節リウマチ、変形性関節症、喘息等を予防、治療又は改善することができる。ただし、本実施形態のヘキソサミニダーゼ遊離抑制剤は、これらの用途以外にもヘキソサミニダーゼ遊離抑制作用を発揮することに意義のあるすべての用途に用いることができる。 The hexosaminidase release inhibitor of this embodiment is a variety of dermatitis (rash), psoriasis, pemphigus vulgaris, atopic dermatitis, and other types of rough skin through the hexosaminidase release inhibitory action of davidigenin. Skin diseases, rheumatoid arthritis, osteoarthritis, asthma and the like can be prevented, treated or ameliorated. However, the hexosaminidase release inhibitor of the present embodiment can be used for all uses other than these uses that are meaningful for exerting the hexosaminidase release inhibitory action.
本実施形態のシクロオキシゲナーゼ−２阻害剤は、ダビジゲニンが有するシクロオキシゲナーゼ−２阻害作用を通じて、接触性皮膚炎（かぶれ）、乾癬、尋常性天疱瘡、アトピー性皮膚炎、その他肌荒れを伴う各種皮膚疾患、関節リウマチ、変形性関節症、喘息等を予防、治療又は改善することができる。ただし、本実施形態のシクロオキシゲナーゼ−２阻害剤は、これらの用途以外にもシクロオキシゲナーゼ−２阻害作用を発揮することに意義のあるすべての用途に用いることができる。 The cyclooxygenase-2 inhibitor according to the present embodiment is capable of contact dermatitis (rash), psoriasis, pemphigus vulgaris, atopic dermatitis, and other various skin diseases accompanied by rough skin, joints, through the cyclooxygenase-2 inhibitory action of davidigenin. It can prevent, treat or ameliorate rheumatism, osteoarthritis, asthma and the like. However, the cyclooxygenase-2 inhibitor of the present embodiment can be used for all uses other than these uses that are meaningful for exerting a cyclooxygenase-2 inhibitory action.
本実施形態の抗炎症剤は、ダビジゲニンが有する腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用及びシクロオキシゲナーゼ−２阻害作用からなる群より選ばれる１種又は２種以上の作用を通じて、各種炎症性疾患、すなわち接触性皮膚炎（かぶれ）、乾癬、尋常性天疱瘡、アトピー性皮膚炎、その他肌荒れを伴う各種皮膚疾患、関節リウマチ、変形性関節症、喘息等を予防・改善することができる。ただし、ダビジゲニンが有する抗炎症作用は、上記作用に基づいて発揮される抗炎症作用に限定されるものではない。 The anti-inflammatory agent of this embodiment is one or more selected from the group consisting of tumor necrosis factor (TNF-α) production inhibitory action, hexosaminidase release inhibitory action and cyclooxygenase-2 inhibitory action of davidigenin. Through action, various inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, atopic dermatitis, other skin diseases with rough skin, rheumatoid arthritis, osteoarthritis, asthma, etc. are prevented / Can be improved. However, the anti-inflammatory action of davidigenin is not limited to the anti-inflammatory action exhibited based on the above action.
また、本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、優れた過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用又は抗炎症作用を有するため、例えば、皮膚外用剤に配合するのに好適である。 Further, the hydrogen peroxide cell disorder preventive / ameliorating agent, glutathione production promoter, antioxidant, tumor tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 of the present embodiment Inhibitors or anti-inflammatory agents are excellent hydrogen peroxide cell disorder preventive / ameliorating action, glutathione production promoting action, antioxidant action, tumor necrosis factor (TNF-α) production inhibiting action, hexosaminidase release inhibiting action, Since it has a cyclooxygenase-2 inhibitory action or an anti-inflammatory action, it is suitable for blending into, for example, a skin external preparation.
ここで、皮膚外用剤としては、その区分に制限はなく、経皮的に使用される皮膚化粧料、医薬部外品、医薬品等を幅広く含むものであり、具体的には、例えば、軟膏、クリーム、乳液、美容液、ローション、パック、ファンデーション、リップクリーム、入浴剤、ヘアートニック、ヘアーローション、石鹸、ボディシャンプー等が挙げられる。 Here, the topical skin preparation is not limited in its category, and includes a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, and the like used transdermally. Creams, milky lotions, beauty essences, lotions, packs, foundations, lip balms, bath salts, hair nicks, hair lotions, soaps, body shampoos and the like.
また、本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、優れた過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用又は抗炎症作用を有するため、例えば、飲食品に配合するのに好適である。飲食品としては、その区分に制限はなく、経口的に摂取される一般食品、健康食品、保健機能食品、医薬部外品、医薬品等を幅広く含むものである。 Further, the hydrogen peroxide cell disorder preventive / ameliorating agent, glutathione production promoter, antioxidant, tumor tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 of the present embodiment Inhibitors or anti-inflammatory agents are excellent hydrogen peroxide cell disorder preventive / ameliorating action, glutathione production promoting action, antioxidant action, tumor necrosis factor (TNF-α) production inhibiting action, hexosaminidase release inhibiting action, Since it has a cyclooxygenase-2 inhibitory action or an anti-inflammatory action, it is suitable for blending into food and drink, for example. The food and drink is not limited in its category, and includes a wide variety of foods such as general foods, health foods, health functional foods, quasi drugs, and pharmaceuticals that are taken orally.
さらに、本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、優れた過酸化水素細胞障害の予防・改善作用、グルタチオン産生促進作用、抗酸化作用、腫瘍壊死因子（ＴＮＦ−α）産生抑制作用、ヘキソサミニダーゼ遊離抑制作用、シクロオキシゲナーゼ−２阻害作用又は抗炎症作用を有するので、線維芽細胞や細胞外マトリックス等に関連する疾患の研究のための試薬としても好適に利用することができる。 Furthermore, the hydrogen peroxide cell disorder preventive / ameliorating agent, glutathione production promoter, antioxidant, tumor tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 of this embodiment Inhibitors or anti-inflammatory agents are excellent hydrogen peroxide cell disorder preventive / ameliorating action, glutathione production promoting action, antioxidant action, tumor necrosis factor (TNF-α) production inhibiting action, hexosaminidase release inhibiting action, Since it has a cyclooxygenase-2 inhibitory action or anti-inflammatory action, it can also be suitably used as a reagent for studying diseases related to fibroblasts, extracellular matrix, and the like.
なお、本実施形態の過酸化水素細胞障害の予防・改善剤、グルタチオン産生促進剤、抗酸化剤、腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤又は抗炎症剤は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物（例えば，マウス，ラット，ハムスター，イヌ、ネコ，ウシ，ブタ，サル等）に対して適用することもできる。 In addition, the preventive / ameliorating agent for hydrogen peroxide cell damage, glutathione production promoter, antioxidant, tumor tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 of this embodiment Inhibitors or anti-inflammatory agents are preferably applied to humans, but as long as their respective effects are exerted, animals other than humans (for example, mice, rats, hamsters, dogs, cats, cattle) , Pigs, monkeys, etc.).
以下、製造例及び試験例を示し、本発明を具体的に説明するが、本発明は下記の各例に何ら制限されるものではない。 Hereinafter, although a manufacture example and a test example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.
〔製造例１〕ダビジゲニンの製造−１
甘草の一種であるグリチルリーザ・インフラータ（Glychyrrhiza inflata）の根茎部を粉砕し、チップ状にした。この甘草チップ１．０ｋｇをフラスコに取り、１０Ｌの５０容量％エタノール水溶液を加え、８０℃で２時間抽出した後、ろ過した。得られたろ液を４０℃以下の温度で減圧下濃縮した後、４０℃で減圧乾燥を行い、黄褐色粉末である甘草根茎部５０％エタノール抽出物（１５０ｇ）を得た。
[Production Example 1] Production of davidigenin-1
The rhizome part of Glychyrrhiza inflata, a kind of licorice, was crushed into chips. 1.0 kg of this licorice chip was placed in a flask, 10 L of 50% by volume aqueous ethanol solution was added, and the mixture was extracted at 80 ° C. for 2 hours, followed by filtration. The obtained filtrate was concentrated under reduced pressure at a temperature of 40 ° C. or lower and then dried under reduced pressure at 40 ° C. to obtain a 50% ethanol extract (150 g) of licorice rhizome as a tan powder.
このようにして得られた甘草根茎部５０％エタノール抽出物１０ｇに水２５ｍＬを加えて懸濁させ、次いでエタノール２５ｍＬおよび５質量％パラジウムカーボンを加えて撹拌し、室温にて懸濁液中に水素ガスを１６時間吹き込んだ。次いで、ろ過により触媒を除去し、ろ液として甘草抽出物の還元処理物（１０ｇ）を得た。 25 mL of water was added to and suspended in 10 g of the 50% ethanol extract of licorice rhizome obtained in this way, and then 25 mL of ethanol and 5% by mass palladium carbon were added and stirred, and hydrogen was added to the suspension at room temperature. Gas was blown for 16 hours. Next, the catalyst was removed by filtration, and a reduced product (10 g) of licorice extract was obtained as a filtrate.
このようにして得られた甘草抽出物の還元処理物１０ｇに９５質量％硫酸５．０ｍＬを加え、８０℃で２時間反応させた。得られた反応液を多孔性樹脂（三菱化学社製，Ｄｉａｉｏｎ ＨＰ−２０，５００ｍＬ）上に付し、水２Ｌ、エタノール２Ｌの順で溶出させた。エタノール２Ｌで溶出させた画分からエタノールを留去し、エタノール溶出画分２．５ｇを得た。このエタノール溶出画分２．５ｇをメタノール：水＝６０：４０（容量比）の混合液に溶解し、ＯＤＳ（富士シリシア化学社製，クロマトレックスＯＤＳ ＤＭ１０２０Ｔ）を充填したガラス製のカラム上部より流入して、ＯＤＳに吸着させた。移動相としてメタノール：水＝６０：４０（容量比）を流し、その溶出液を集め、溶媒を留去し、ダビジゲニン濃縮液３００ｍｇを得た。得られたダビジゲニン濃縮液を、下記の条件で液体クロマトグラフィーを用いて分画した。 To 10 g of the reduced product of the licorice extract thus obtained, 5.0 mL of 95% by mass sulfuric acid was added and reacted at 80 ° C. for 2 hours. The obtained reaction solution was applied onto a porous resin (Diaion HP-20, 500 mL, manufactured by Mitsubishi Chemical Corporation) and eluted in the order of 2 L of water and 2 L of ethanol. Ethanol was distilled off from the fraction eluted with 2 L of ethanol to obtain 2.5 g of ethanol-eluted fraction. Dissolve 2.5 g of this ethanol-eluted fraction in a mixed solution of methanol: water = 60: 40 (volume ratio), and flow from the top of a glass column packed with ODS (Fuji Silysia Chemical Co., Ltd., Chromatrex ODS DM1020T). And it was made to adsorb | suck to ODS. Methanol: water = 60: 40 (volume ratio) was passed as a mobile phase, the eluate was collected, the solvent was distilled off, and 300 mg of a davidigenin concentrate was obtained. The obtained davidigenin concentrate was fractionated using liquid chromatography under the following conditions.
＜液体クロマトグラフィー条件＞
固定相：ＪＡＩＧＥＬ ＧＳ−３１０（日本分析工業社製）
カラム径：２０ｍｍ
カラム長：２５０ｍｍ
移動相流量：５ｍＬ／ｍｉｎ
検出：ＲＩ
<Liquid chromatography conditions>
Stationary phase: JAIGEL GS-310 (manufactured by Nippon Analytical Industrial Co., Ltd.)
Column diameter: 20mm
Column length: 250mm
Mobile phase flow rate: 5 mL / min
Detection: RI
ここで、保持時間４０分〜５０分に溶出する画分をリサイクルＨＰＬＣにより精製を行い、精製物を得た（２２０ｍｇ）。得られた精製物について、１３Ｃ−ＮＭＲにより分析した結果を以下に示す。 Here, the fraction eluted at a retention time of 40 to 50 minutes was purified by recycle HPLC to obtain a purified product (220 mg). About the obtained refined | purified substance, the result analyzed by < 13 > C-NMR is shown below.
＜１３Ｃ−ＮＭＲケミカルシフトδ（帰属炭素）＞
２０５．６（Ｃ＝Ｏ），１６６．４（４’−Ｃ），１６６．３（２’−Ｃ），１５６．７（４−Ｃ），１３３．７（６’−Ｃ），１３３．２（１−Ｃ），１３０．４（２，５−Ｃ），１１６．２（３，５−Ｃ），１１４．１（１’−Ｃ），１０９．１（５’−Ｃ），１０３．７（３’−Ｃ），４０．９（α−Ｃ），３１．０（β−Ｃ）
< 13 C-NMR chemical shift δ (assigned carbon)>
205.6 (C = O), 166.4 (4'-C), 166.3 (2'-C), 156.7 (4-C), 133.7 (6'-C), 133. 2 (1-C), 130.4 (2,5-C), 116.2 (3,5-C), 114.1 (1′-C), 109.1 (5′-C), 103 .7 (3′-C), 40.9 (α-C), 31.0 (β-C)
以上の結果から、甘草抽出物の還元処理物を多孔性樹脂により分画し、ＯＤＳにより分離し、さらに液体クロマトグラフィーにより精製して得られた精製物が、下記式で表されるダビジゲニン（試料１）であることが確認された。 From the above results, the purified product obtained by fractionating the reduction-treated product of licorice extract with a porous resin, separating it with ODS, and further purifying it with liquid chromatography is a davidigenin (sample) represented by the following formula: 1) was confirmed.
〔製造例２〕ダビジゲニンの製造−２
４８％質量水酸化ナトリウム水溶液１０ｍＬにイオン交換水１０ｍＬを加えて希釈した。これを６０℃に加熱し、撹拌しながらｐ−ヒドロキシベンズアルデヒド１．２ｇを加えて溶解し、次いで２’，４’−ジヒドロキシアセトフェノン１．５ｇを加えて溶解した。この反応液を６０℃に加熱したまま２４時間撹拌し、さらに加熱を止めて室温で４８時間撹拌した。得られた反応液を氷浴上で冷却しながら、水８０ｍＬを用いて希釈した後、あらかじめ調製した１０％硫酸水溶液４０ｍＬを加えて黄色結晶を析出させた。この懸濁液からろ紙を用いたろ過によって沈殿部をろ取した。ろ取した沈殿を４０℃にて減圧乾燥し、２’，４，４’−トリヒドロキシカルコン５００ｍｇを黄色結晶として得た。
[Production Example 2] Production of dabidigenin-2
10 mL of ion exchange water was added to 10 mL of a 48% mass aqueous sodium hydroxide solution for dilution. This was heated to 60 ° C. and 1.2 g of p-hydroxybenzaldehyde was added and dissolved while stirring, and then 1.5 g of 2 ′, 4′-dihydroxyacetophenone was added and dissolved. The reaction solution was stirred for 24 hours while being heated to 60 ° C., and then the heating was stopped and the mixture was stirred at room temperature for 48 hours. While cooling the obtained reaction liquid on an ice bath, it diluted with 80 mL of water, Then, 40 mL of 10% sulfuric acid aqueous solution prepared previously was added, and the yellow crystal | crystallization was deposited. The precipitate was collected from the suspension by filtration using filter paper. The precipitate collected by filtration was dried under reduced pressure at 40 ° C. to obtain 500 mg of 2 ′, 4,4′-trihydroxychalcone as yellow crystals.
上記の反応を繰り返すことで得られた２’，４，４’−トリヒドロキシカルコン１０ｇを、密封可能な反応容器内でエタノール５０ｍＬに溶解し、５質量％パラジウムカーボン１．０ｇを加えた。この懸濁液を激しく撹拌しながら、容器内を減圧しては水素ガスを吹き込む操作を数回繰り返し、容器内を水素ガスで充満させた。反応液を４時間激しく撹拌した後に、ろ過によって触媒を除去した。得られたろ液に水１００ｍＬを加えた。析出した白色結晶をろ紙ろ過によってろ取し、これを４０℃にて減圧乾燥することで白色結晶を得た（９．５ｇ）。 10 g of 2 ', 4,4'-trihydroxychalcone obtained by repeating the above reaction was dissolved in 50 mL of ethanol in a sealable reaction vessel, and 1.0 g of 5 mass% palladium carbon was added. While the suspension was vigorously stirred, the operation of reducing the pressure in the container and blowing in hydrogen gas was repeated several times to fill the container with hydrogen gas. After the reaction solution was vigorously stirred for 4 hours, the catalyst was removed by filtration. 100 mL of water was added to the obtained filtrate. The precipitated white crystals were collected by filtration with a filter paper and dried under reduced pressure at 40 ° C. to obtain white crystals (9.5 g).
得られた白色結晶を１３Ｃ−ＮＭＲにより分析したところ、試料１の結果と一致し、得られた白色結晶がダビジゲニンであることが確認された。 When the obtained white crystals were analyzed by 13 C-NMR, it was confirmed that the obtained white crystals were davidigenin, which coincided with the result of Sample 1.
〔試験例１〕過酸化水素細胞障害の予防・改善試験
製造例１で得られたダビジゲニン（試料１）について、下記の試験方法により過酸化水素細胞障害の予防・改善作用を試験した。
[Test Example 1] Prevention / Improvement Test of Hydrogen Peroxide Cell Damage The davidigenin (sample 1) obtained in Production Example 1 was tested for the prevention / amelioration action of hydrogen peroxide cell damage by the following test method.
ヒト正常皮膚線維芽細胞（ＮＢ１ＲＧＢ）を、１０％ＦＢＳ含有α−ＭＥＭ培地を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を２．５×１０５ｃｅｌｌｓ／ｍＬの細胞濃度になるように５％ＦＢＳ含有α−ＭＥＭ培地で希釈した後、４８ウェルのマイクロプレートに１ウェル当たり２００μＬずつ播種し、一晩培養した。 Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells are diluted with α-MEM medium containing 5% FBS so that the cell concentration becomes 2.5 × 10 5 cells / mL, then seeded at 200 μL per well in a 48-well microplate, and cultured overnight. did.
培養後、培養液を除去し、１％ＦＢＳ含有α−ＭＥＭ培地で溶解した被験試料（試料１，試料濃度は下記表１を参照）を、各ウェルに２００μＬずつ添加し、２４時間培養した。培養終了後、各ウェルから培地を抜き、４００μＬのＰＢＳ緩衝液で洗浄し、過酸化水素を溶解したＨａｎｋ’ｓ緩衝液（過酸化水素最終濃度：１ｍＭ）を２００μＬ添加し、２時間培養した。 After culturing, the culture solution was removed, and a test sample dissolved in 1% FBS-containing α-MEM medium (sample 1, refer to Table 1 below for sample concentration) was added to each well in an amount of 200 μL and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS buffer, and 200 μL of Hank's buffer (hydrogen peroxide final concentration: 1 mM) dissolved in hydrogen peroxide was added and cultured for 2 hours.
また、過酸化水素を溶解していないＨａｎｋ’ｓ緩衝液を２００μＬ添加し、同様の条件で培養した。さらに、試料溶液を添加せずに培養し、培養後、過酸化水素を溶解したＨａｎｋ’ｓ緩衝液を２００μＬ添加し、同様の条件で培養した。 In addition, 200 μL of Hank's buffer solution in which hydrogen peroxide was not dissolved was added and cultured under the same conditions. Furthermore, it culture | cultivated without adding a sample solution, and after culture | cultivation, 200 microliters of Hank's buffer solution which melt | dissolved hydrogen peroxide was added, and it culture | cultivated on the same conditions.
培養後、４００μＬのＰＢＳ緩衝液で洗浄し、１％ＦＢＳ含有α−ＭＥＭ培地で溶解した０．０５ｍｇ／ｍＬのニュートラルレッド溶液を、２００μＬずつ添加し、２．５時間培養した。この後、ニュートラルレッド溶液を除去し、エタノール・酢酸溶液（エタノール：酢酸：水＝５０：１：４９）３００μＬを各ウェルに加え、色素を抽出した。その後、マイクロプレートリーダーを用い５４０ｎｍでの吸光度を測定し、過酸化水素障害抑制率（％）を求めた。なお、過酸化水素障害抑制率は、下記の計算式により算出した。 After culturing, 200 μL of 0.05 mg / mL neutral red solution dissolved in 400 μL PBS buffer and dissolved in α-MEM medium containing 1% FBS was added and cultured for 2.5 hours. Thereafter, the neutral red solution was removed, and 300 μL of an ethanol / acetic acid solution (ethanol: acetic acid: water = 50: 1: 49) was added to each well to extract the dye. Thereafter, the absorbance at 540 nm was measured using a microplate reader to determine the hydrogen peroxide disorder inhibition rate (%). In addition, the hydrogen peroxide disorder inhibition rate was calculated by the following formula.
過酸化水素障害抑制率(％)＝{１−(Ｃ−Ａ)}／(Ｃ−Ｂ)×１００
上記式において、Ａは「被験試料添加・過酸化水素処理」の吸光度を、Ｂは「試料無添加・過酸化水素処理」の吸光度を、Ｃは「試料無添加・過酸化水素無処理」の吸光度を、示す。
上記試験の結果を表１に示す。
Hydrogen peroxide damage inhibition rate (%) = {1- (C−A)} / (C−B) × 100
In the above formula, A is the absorbance of “test sample addition / hydrogen peroxide treatment”, B is the absorbance of “sample no addition / hydrogen peroxide treatment”, and C is “sample no addition / hydrogen peroxide treatment”. Absorbance is shown.
The results of the above test are shown in Table 1.
表１に示すように、ダビジゲニン（試料１）は、高い過酸化水素障害抑制率を示した。この結果から、ダビジゲニンは、優れた過酸化水素細胞障害の予防・改善作用を有することが確認された。 As shown in Table 1, davidigenin (sample 1) showed a high hydrogen peroxide damage inhibition rate. From this result, it was confirmed that davidigenin has an excellent action for preventing and improving hydrogen peroxide cell damage.
〔試験例２〕グルタチオン産生促進作用試験
製造例１により得られたダビジゲニン（試料１）について、以下のようにしてグルタチオン産生促進作用を試験した。
[Test Example 2] Glutathione production promoting action test With respect to davidigenin (sample 1) obtained in Production Example 1, the glutathione production promoting action was tested as follows.
ヒト正常皮膚線維芽細胞（ＮＢ１ＲＧＢ）を、１０％ＦＢＳ含有α−ＭＥＭ培地を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を２．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるように１０％ＦＢＳ含有α−ＭＥＭ培地で希釈した後、４８ウェルプレートに１ウェル当たり２００μＬずつ播種し、一晩培養した。 Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with 10% FBS-containing α-MEM medium to a cell density of 2.0 × 10 5 cells / mL, then seeded at 200 μL per well in a 48-well plate and cultured overnight.
培養後、１％ＦＢＳ含有α−ＭＥＭ培地で溶解した被験試料（試料１，試料濃度は下記表２を参照）を各ウェルに２００μＬ添加し、２４時間培養した。培養終了後、各ウェルから培地を抜き、４００μＬのＰＢＳ緩衝液にて洗浄後、１５０μＬのＭ−ＰＥＲ（ＰＩＥＲＣＥ社製）を使用して細胞を溶解した。 After culturing, 200 μL of a test sample dissolved in 1% FBS-containing α-MEM medium (Sample 1, see Table 2 below for sample concentration) was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS buffer, and the cells were lysed using 150 μL of M-PER (PIERCE).
このうちの１００μＬを使用して総グルタチオンの定量を行った。すなわち、９６ウェルプレートに溶解した細胞抽出液１００μＬ、０．１Ｍのリン酸緩衝液５０μＬ、２ｍＭのＮＡＤＰＨ２５μＬ及びグルタチオンレダクターゼ２５μＬ（終濃度１７．５ｕｎｉｔ／ｍＬ）を加え３７℃で１０分間加温した後、１０ｍＭの5,5'-dithiobis(2-nitrobenzoic acid)２５μＬを加え、５分後までの波長４１２ｎｍにおける吸光度を測定し、ΔＯＤ／ｍｉｎを求めた。総グルタチオン濃度は、酸化型グルタチオン（和光純薬社製）を使用して作成した検量線をもとに算出した。得られた値を総タンパク量当たりのグルタチオン量に補正した後、下記式によりグルタチオン産生促進率（％）を算出した。 Of these, 100 μL was used to quantify total glutathione. That is, after adding 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 units / mL), the mixture was heated at 37 ° C. for 10 minutes. 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated by the following formula.
グルタチオン産生促進率(％)＝Ｂ／Ａ×１００
式中、Ａは「試料無添加時の細胞中における総タンパク量当たりのグルタチオン量（対照）」を表し、Ｂは「被験試料添加時の細胞中における総タンパク量当たりのグルタチオン量」を表す。
結果を表２に示す。
Glutathione production promotion rate (%) = B / A × 100
In the formula, A represents “the amount of glutathione per total protein in the cell when no sample is added (control)”, and B represents “the amount of glutathione per total amount of protein in the cell when added to the test sample”.
The results are shown in Table 2.
表２に示すように、ダビジゲニン（試料１）は優れたグルタチオン産生促進作用を有することが確認された。 As shown in Table 2, it was confirmed that davidigenin (sample 1) has an excellent glutathione production promoting action.
〔試験例３〕ＴＮＦ−α産生抑制作用試験
製造例１により得られたダビジゲニン（試料１）について、以下のようにしてＴＮＦ−α産生抑制作用を試験した。
[Test Example 3] TNF-α production inhibitory action test The davidigenin (sample 1) obtained in Production Example 1 was tested for TNF-α production inhibitory action as follows.
マウスマクロファージ細胞（ＲＡＷ２６４．７）を、１０％ＦＢＳ含有ダルベッコＭＥＭ培地を用いて培養した後、セルスクレーパーにより細胞を回収した。回収した細胞を１．０×１０６ｃｅｌｌｓ／ｍＬの細胞密度になるように１０％ＦＢＳ含有ダルベッコＭＥＭ培地で希釈した後、９６ウェルプレートに１ウェル当たり１００μＬずつ播種し、４時間培養した。 After culturing mouse macrophage cells (RAW264.7) using Dulbecco's MEM medium containing 10% FBS, the cells were collected with a cell scraper. The collected cells were diluted with Dulbecco's MEM medium containing 10% FBS to a cell density of 1.0 × 10 6 cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured for 4 hours.
培養終了後、培地を抜き、終濃度２％ＤＭＳＯを含む１０％ＦＢＳ含有ダルベッコＭＥＭ培地で溶解した被験試料（試料１，試料濃度は下記表３を参照）を各ウェルに１００μＬずつ添加し、終濃度１μｇ／ｍＬで１０％ＦＢＳ含有ダルベッコＭＥＭ培地に溶解したリポポリサッカライド（DIFCO社製，ＬＰＳ，E.coli0111;B4）を１００μＬ加え、２４時間培養した。培養終了後、各ウェルの培養上清中のＴＮＦ−α量を、サンドイッチＥＬＩＳＡ法を用いて測定し、測定結果から下記式によりＴＮＦ−α産生抑制率（％）を算出した。 After completion of the culture, the medium was removed, and 100 μL of a test sample dissolved in Dulbecco's MEM medium containing 10% FBS containing a final concentration of 2% DMSO (Sample 1, see Table 3 below) was added to each well. 100 μL of lipopolysaccharide (DIFCO, LPS, E. coli 0111; B4) dissolved in Dulbecco's MEM medium containing 10% FBS at a concentration of 1 μg / mL was added and cultured for 24 hours. After completion of the culture, the amount of TNF-α in the culture supernatant of each well was measured using a sandwich ELISA method, and the TNF-α production inhibition rate (%) was calculated from the measurement result according to the following formula.
ＴＮＦ−α産生抑制率(％)＝{(Ｂ−Ａ)／Ｂ}×１００
式中、Ａは「被験試料添加時のＴＮＦ−α量」を表し、Ｂは「試料無添加時のＴＮＦ−α量」を表す。
結果を表３に示す。
TNF-α production inhibition rate (%) = {(B−A) / B} × 100
In the formula, A represents “the amount of TNF-α when a test sample is added”, and B represents the “the amount of TNF-α when no sample is added”.
The results are shown in Table 3.
表３に示すように、ダビジゲニン（試料１）は、優れたＴＮＦ−α産生抑制作用を有することが確認された。 As shown in Table 3, it was confirmed that davidigenin (sample 1) has an excellent TNF-α production inhibitory action.
〔試験例４〕ヘキソサミニダーゼ遊離抑制作用試験
製造例１により得られたダビジゲニン（試料１）について、以下のようにしてヘキソサミニダーゼ遊離抑制作用を試験した。
[Test Example 4] Hexosaminidase release inhibitory action test The davidigenin (sample 1) obtained in Production Example 1 was tested for hexosaminidase release inhibitory action as follows.
ラット好塩基球白血病細胞（ＲＢＬ−２Ｈ３）を１５％ＦＢＳ添加Ｓ−ＭＥＭ培地を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を４．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度となるように１５％ＦＢＳ添加Ｓ−ＭＥＭ培地で希釈し、終濃度０．５μｇ／ｍＬとなるようにDNP-specific IgEを添加した後、９６ウェルプレートに１ウェルあたり１００μＬずつ播種し、一晩培養した。 Rat basophil leukemia cells (RBL-2H3) were cultured in S-MEM medium supplemented with 15% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with S-MEM medium supplemented with 15% FBS so that the cell density was 4.0 × 10 5 cells / mL, and DNP-specific IgE was added so that the final concentration was 0.5 μg / mL. Thereafter, 100 μL per well was seeded in a 96-well plate and cultured overnight.
培養終了後、培地を抜き、シリガリアン緩衝液５００μＬにて洗浄を２回行った。次に、同緩衝液３０μＬ及び同緩衝液にて調製した被験試料（試料１，試料濃度は下記表４を参照）を１０μＬ加え、３７℃にて１０分間静置した。その後、１００ｎｇ／ｍＬのＤＮＰ−ＢＳＡ溶液１０μＬを加え、３７℃にて１５分間静置し、ヘキソサミニダーゼを遊離させた。 After completion of the culture, the medium was removed and washed twice with 500 μL of Siligalian buffer. Next, 10 μL of the same buffer solution (30 μL) and a test sample prepared with the same buffer solution (Sample 1, see Table 4 below for sample concentration) were added and allowed to stand at 37 ° C. for 10 minutes. Thereafter, 10 μL of a 100 ng / mL DNP-BSA solution was added, and the mixture was allowed to stand at 37 ° C. for 15 minutes to release hexosaminidase.
その後、９６ウェルプレートを氷上に静置することにより遊離を停止した。各ウェルの細胞上清１０μＬ及び１ｍＭのｐ−ＮＡＧ（ｐ−ニトロフェニル−Ｎ−アセチル−β−Ｄ−グルコサミニド）溶液１０μＬを、新たな９６ウェルプレートに添加し、３７℃で１時間反応させた。 Thereafter, the release was stopped by allowing the 96-well plate to stand on ice. 10 μL of cell supernatant in each well and 10 μL of 1 mM p-NAG (p-nitrophenyl-N-acetyl-β-D-glucosaminide) solution were added to a new 96-well plate and reacted at 37 ° C. for 1 hour. .
反応終了後、各ウェルに０．１ＭのＮａ２ＣＯ３／ＮａＨＣＯ３２５０μＬを加え、波長４１５ｎｍにおける吸光度を測定した。また、空試験として、細胞上清１０μＬと、０．１ＭのＮａ２ＣＯ３／ＮａＨＣＯ３２５０μＬとの混合液の波長４１５ｎｍにおける吸光度を測定し、補正した。得られた測定結果から、下記式によりヘキソサミニダーゼ遊離抑制率（％）を算出した。 After completion of the reaction, 250 μL of 0.1 M Na 2 CO 3 / NaHCO 3 was added to each well, and the absorbance at a wavelength of 415 nm was measured. As a blank test, the absorbance at a wavelength of 415 nm of a mixed solution of 10 μL of cell supernatant and 250 μL of 0.1 M Na 2 CO 3 / NaHCO 3 was measured and corrected. From the obtained measurement results, the hexosaminidase release inhibition rate (%) was calculated by the following formula.
ヘキソサミニダーゼ遊離抑制率(％)＝{１−(Ｂ−Ｃ)／Ａ}×１００
式中、Ａは「試料無添加での波長４１５ｎｍにおける吸光度」を表し、Ｂは「被験試料添加での波長４１５ｎｍにおける吸光度」を表し、Ｃは「被験試料添加・ｐ−ＮＡＧ無添加での波長４１５ｎｍにおける吸光度」を表す。
結果を表４に示す。
Inhibition rate of hexosaminidase release (%) = {1− (B−C) / A} × 100
In the formula, A represents “absorbance at a wavelength of 415 nm without addition of a sample”, B represents “absorbance at a wavelength of 415 nm with addition of a test sample”, and C represents “wavelength without addition of a test sample / no p-NAG”. "Absorbance at 415 nm".
The results are shown in Table 4.
表４に示す結果から、ダビジゲニン（試料１）は、優れたヘキソサミニダーゼ遊離抑制作用を有することが確認された。また、ヘキソサミニダーゼ遊離抑制作用の程度は、ダビジゲニンの濃度によって調節できることが確認された。 From the results shown in Table 4, it was confirmed that davidigenin (sample 1) has an excellent hexosaminidase release inhibitory action. It was also confirmed that the degree of hexosaminidase release inhibitory action can be adjusted by the concentration of davidigenin.
〔試験例５〕シクロオキシゲナーゼ−２（ＣＯＸ−２）活性阻害作用試験
製造例１で得られたダビジゲニン（試料１）について、以下のようにしてシクロオキシゲナーゼ−２活性阻害作用を試験した。
[Test Example 5] Cyclooxygenase-2 (COX-2) activity inhibitory action test The davidigenin (sample 1) obtained in Production Example 1 was tested for cyclooxygenase-2 activity inhibitory action as follows.
マウスマクロファージ細胞（ＲＡＷ２６４．７）を、１０％ＦＢＳ含有ダルベッコＭＥＭ培地を用いて培養した後、セルスクレーパーにより細胞を回収した。回収した細胞を２．０×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるように１０％ＦＢＳ含有ダルベッコＭＥＭ培地で希釈した後、９６ウェルプレートに１ウェルあたり１００μＬずつ播種し、１８時間培養した。 After culturing mouse macrophage cells (RAW264.7) using Dulbecco's MEM medium containing 10% FBS, the cells were collected with a cell scraper. The collected cells were diluted with Dulbecco's MEM medium containing 10% FBS to a cell density of 2.0 × 10 5 cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured for 18 hours.
培養終了後、すでに存在するＣＯＸ−１及び少量発現しているＣＯＸ−２をアセチル化して失活させるため、培地を５００μｍｏｌ／Ｌアスピリン含有培地に交換し、４時間培養した。細胞をＰＢＳ緩衝液で３回洗浄し、終濃度０．５％ＤＭＳＯを含む１０％ＦＢＳ含有ダルベッコＭＥＭ培地で溶解した被験試料（試料１，試料濃度は下記表５を参照）を各ウェルに１５０μＬずつ添加した後、終濃度１μｇ／ｍＬで１０％ＦＢＳ含有ダルベッコＭＥＭ培地に溶解したリポポリサッカライド（DIFCO社製，ＬＰＳ，E.coli 0111；B4）を５０μＬ添加し、２４時間培養した。 After culturing, in order to acetylate and inactivate COX-1 already present and COX-2 expressed in a small amount, the medium was changed to a medium containing 500 μmol / L aspirin and cultured for 4 hours. 150 μL of a test sample (sample 1, refer to Table 5 below for sample concentration) in which each cell was washed 3 times with PBS buffer and lysed in Dulbecco's MEM medium containing 10% FBS containing 0.5% final DMSO After each addition, 50 μL of lipopolysaccharide (DIFCO, LPS, E. coli 0111; B4) dissolved in Dulbecco's MEM medium containing 10% FBS at a final concentration of 1 μg / mL was added and cultured for 24 hours.
培養終了後、各ウェルの培養上清中のプロスタグランジンＥ２（ＰＧＥ２）量を、ＰＧＥ２ ＥＩＡ Ｋｉｔ（Cayman Chemical社製）を用いて定量した。定量結果から、下記式によりシクロオキシゲナーゼ−２活性阻害率（％）を算出した。 After completion of the culture, the amount of prostaglandin E 2 (PGE 2 ) in the culture supernatant of each well was quantified using PGE 2 EIA Kit (Cayman Chemical). From the quantification result, cyclooxygenase-2 activity inhibition rate (%) was calculated by the following formula.
ＣＯＸ−２活性阻害率(％)＝{１−(Ａ−Ｃ)／(Ｂ−Ｃ)}×１００
式中、Ａは「被験試料添加・ＬＰＳ刺激時のＰＧＥ２量」を表し、Ｂは「試料無添加・ＬＰＳ刺激時のＰＧＥ２量」を表し、Ｃは「試料無添加・ＬＰＳ無刺激時のＰＧＥ２量」を表す。
結果を表５に示す。
COX-2 activity inhibition rate (%) = {1− (AC) / (BC)} × 100
In the formula, A represents "the amount of PGE 2 at the test specimen · LPS stimulation", B represents "the amount of PGE 2 at the sample without additives · LPS stimulation", when C is "no sample added · LPS non-stimulated Of PGE 2 ”.
The results are shown in Table 5.
表５に示すように、ダビジゲニン（試料１）は、シクロオキシゲナーゼ−２活性阻害作用を有することが確認された。また、シクロオキシゲナーゼ−２活性阻害作用の程度は、ダビジゲニンの濃度によって調節できることが確認された。 As shown in Table 5, davidigenin (sample 1) was confirmed to have a cyclooxygenase-2 activity inhibitory action. Moreover, it was confirmed that the degree of cyclooxygenase-2 activity inhibitory action can be adjusted by the concentration of davidigenin.
本発明のグルタチオン産生促進剤、過酸化水素細胞障害の予防・改善剤及び抗酸化剤は、酸化ストレスにより誘発される各種疾患の予防又は改善に大きく貢献できる。また、腫腫瘍壊死因子（ＴＮＦ−α）産生抑制剤、ヘキソサミニダーゼ遊離抑制剤、シクロオキシゲナーゼ−２阻害剤及び抗炎症剤は、各種炎症性疾患の予防又は改善に大きく貢献できる。 The glutathione production promoter, the hydrogen peroxide cell disorder preventive / ameliorating agent and the antioxidant of the present invention can greatly contribute to the prevention or improvement of various diseases induced by oxidative stress. Moreover, tumor tumor necrosis factor (TNF-α) production inhibitor, hexosaminidase release inhibitor, cyclooxygenase-2 inhibitor and anti-inflammatory agent can greatly contribute to the prevention or improvement of various inflammatory diseases.
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